Chemical engineering processes and apparatus for the synthesis of compounds

ABSTRACT

The present invention provides methods for producing cannabinoids and cannabinoid analogs as well as a system for producing these compounds. The inventive method is directed to contacting a compound according to Formula I or Formula II with a cannabinoid synthase. 
     
       
         
         
             
             
         
       
     
     Also described is a system for producing cannabinoids and cannabinoid analogs by contacting a THCA synthase with a cannabinoid precursor and modifying at least one property of the reaction mixture to influence the quantity formed of a first cannabinoid relative to the quantity formed of a second cannabinoid.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/171,517, filed Jun. 2, 2016, incorporated herein by reference in itsentirety, which is a Continuation of U.S. application Ser. No.14/836,339, filed Aug. 26, 2015, now U.S. Pat. No. 9,359,625 issued Jun.7, 2016, incorporated herein by reference in its entirety, which is aContinuation of US Application PCT/US2014/018944, filed Feb. 27, 2014,incorporated herein by reference in its entirety, which claims priorityfrom Provisional Application U.S. Application 61/770,766, filed Feb. 28,2013, incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to biosynthesis of cannabinoids. Morespecifically, this invention relates to methods for using protein kinasesynthase enzymes, responsible for the synthesis of cannabinoids inplants, to manufacture ex vivo milligram to gram or kilogram quantitiesof cannabinoids suitable for pharmaceutical and nutraceuticalapplications.

BACKGROUND OF THE INVENTION

Cannabinoids are compounds derived from Cannabis sativa, an annual plantin the Cannabaceae family. The plant contains about 60 cannabinoids. Themost well-known naturally occurring cannabinoid is tetrahydrocannabinol(THC), which is used for the treatment of a wide range of medicalconditions, including glaucoma, AIDS wasting, neuropathic pain,treatment of spasticity associated with multiple sclerosis, fibromyalgiaand chemotherapy-induced nausea. Additionally, THC has been reported toexhibit a therapeutic effect in the treatment of allergies,inflammation, infection, epilepsy, depression, migraine, bipolardisorders, anxiety disorder, and drug dependency and withdrawalsyndromes. THC is particularly effective as an anti-emetic drug and isadministered to curb emesis, a common side effect accompanying the useof opioid analgesics and anesthetics, highly active anti-retroviraltherapy and cancer chemotherapy.

Cannabinoids are increasingly being used for pharmaceutical andnutraceutical applications. Cannabinoid compounds used in suchapplications are almost exclusively obtained from natural sources, forexample, from plant tissue. Thus, the prior art discloses obtainingcannabinoid compounds from the trichomes of the C. sativa plant usingdifferent solvent extraction methodologies. Some draw backs associatedwith such methods include poor yields, high costs associated withgrowing and maintenance of the plant and costs associated withextraction and purification of the plant extract. Security of plants isalso an important consideration that adds to the cost of producingpharmaceutical grade cannabinoid compounds.

However, the increasing importance of cannabinoid compounds,particularly, for treating nausea and vomiting associated withchemotherapy, pain, as agents for stimulating appetite in AIDS patientssuffering from the wasting syndrome and for treatment of glaucoma haveprompted the present inventors to develop an ex vivo enzyme catalyzedsemi-synthetic protocol for the large scale production of cannabinoidsand cannabinoid analogs. The inventive methodologies also permit thesynthesis of cannabinoids and their analogs at reduced costs.

SUMMARY OF THE INVENTION

The foregoing general description and following brief description of thedrawings and the detailed description are exemplary and explanatory andare intended to provide further explanation of the invention as claimed.Other objects, advantages, and novel features will be readily apparentto those skilled in the art from the following detailed description ofthe invention.

In one of its aspects the present invention provides a method ofproducing a cannabinoid or a cannabinoid analog by:

-   (a) selecting a compound according to Formula I;

-   (b) selecting a cannabinoid synthase as a catalyst for transforming    the Formula I compound to the cannabinoid or cannabinoid analog;-   (c) contacting the Formula I compound with the cannabinoid synthase;    and-   (d) isolating the product from step (b). Pursuant to this synthetic    strategy, the isolated product may be decarboxylated.

In Formula I, R is selected from —OH, halogen, —SH, or a —NR_(a)R_(b)group. Substituents R₁ and R₂ are each independently selected from thegroup consisting of —H, —C(O)R_(a), —OR_(a), an optionally substitutedC₁-C₁₀ linear or branched alkylene, an optionally substituted C₂-C₁₀linear or branched alkenylene, an optionally substituted C₂-C₁₀ linearor branched alkynylene, an optionally substituted C₃-C₁₀ aryl, anoptionally substituted C₃-C₁₀ cycloalkyl,(C₃-C₁₀)aryl-(C_(i)-C₁₀)alkylene, (C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and(C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene.

For certain Formula I compounds, R₁ and R₂ together with the carbonatoms to which they are bonded form a C₅-C₁₀ cyclic ring. In one aspect,the C₅-C₁₀ cyclic ring comprises one or more heteroatoms selected fromoxygen, sulfur or nitrogen. R₃ in Formula I is selected from the groupconsisting of H, —C(O)R_(a) and C₁-C₁₀ linear or branched alkyl, whileR_(a) and R_(b) are each independently —H, —OH, —SH, —NH₂, (C₁-C₁₀)linear or branched alkyl, or a C₃-C₁₀ cycloalkyl. The synthesis oftetrahydrocannabinol or cannabidiol, however, are not permitted by theinventive method.

Pursuant to this strategy, the cannabinoid synthase is selected from thegroup cannabidiolic acid synthase, a tetrahydrocannabinolic acidsynthase or a cannabichromene acid synthase.

R2 in Formula I can be a linear alkylene, a C₂-C₁₀ alkenylene, such as

or a C₂-C₁₀ linear or branched alkynylene selected from

For certain Formula I compounds R₂ is

where X is selected from —OH, —SH, or —NR_(a)R_(b).

In one of its aspects, the inventive method describes the immobilizationof a cannabinoid synthase to a solid support. Cannabinoids andcannabinoid analogs produced according to the method described aboveexists as a single enantiomer. The enantiomeric purity of thesecompounds is from at least 95% to at least 99%.

According to another embodiment is provided a method of producing acannabinoid or a cannabinoid analog according to Formula II, by:

(a) reacting a compound according to Formula III with a compoundaccording to Formula IV;

in the presence of an enzyme that catalyzes the reaction of the FormulaIII and Formula IV compounds to form a Formula II compound;

(b) contacting the Formula II compound with a cannabinoid synthase;

(c) isolating the product from step (b); and

(d) optionally decarboxylating the product from (c) to obtain thecannabinoid or the cannabinoid analog.

Substituents R, R₁, R₂ and R₃ are as defined above and R₅ is selectedfrom the group consisting of a linear or branched (C₁-C₁₀)alkylene, alinear or branched (C₂-C₁₀)alkenylene, a linear or branched(C₂-C₁₀)alkynylene, —C(O)—(C₁-C₁₀)alkylene, —C(O)—(C₂-C₁₀)alkenylene and—C(O)—(C₂-C₁₀)alkynylene. Furthermore, for Formulae II, III and IVcompounds, any alkylene, alkenylene, alkynylene, aryl, arylalkylene, orcycloalkyl group is further substituted with one or more groups selectedfrom the group consisting of —OH, halogen, —NR_(b)R_(c), —C(O)R_(a),—C(O)NR_(b)R_(c), (C₁-C₁₀)alkyl, —CN, (C₁-C₄)alkoxy, (C₁-C₄)haloalkyl,and (C₁-C₄)hydroxyalkyl and R_(a), R_(b) and R_(c) can eachindependently be —H, —OH, —SH, —NH₂, (C₁-C₁₀) linear or branched alkyl,or a C₃-C₁₀ cycloalkyl.

Also provided is a method for producing a tetrahydrocannabinol,cannabichrome, or both tetrahydrocannabinol and cannabichrome, or theiranalogs by:

(a) selecting a compound according to Formula V;

(b) contacting the Formula V compound with tetrahydrocannabinolic acidsynthase;

(c) modifying at least one property of a reaction mixture comprising aFormula V compound and tetrahydrocannabinolic acid synthase to obtain atetrahydrocannabinol, a cannabichromene, or both tetrahydrocannabinoland cannabichrome, or their analogs as products.

For Formula V compounds, R is selected from —OH, halogen, —SH, or a—NR_(a)R_(b) group, R₁ and R₂ are each independently selected from thegroup consisting of —H, —C(O)R_(a), —OR_(a), an optionally substitutedC₁-C₁₀ linear or branched alkylene, an optionally substituted C₂-C₁₀linear or branched alkenylene, an optionally substituted C₂-C₁₀ linearor branched alkynylene, an optionally substituted C₃-C₁₀ aryl, anoptionally substituted C₃-C₁₀ cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene,and R₃ is selected from H, —C(O)R_(a), or C₁-C₁₀ linear or branchedalkyl. For certain compounds, R₁ and R₂ together with the carbon atomsto which they are bonded form a C₅-C₁₀ cyclic ring and substituentgroups R_(a) and R_(b) are each independently —H, —OH, —SH, —NH₂,(C₁-C₁₀) linear or branched alkyl, or a C₃-C₁₀ cycloalkyl.

In one aspect, modifying at least one property of a reaction mixturecomprises changing the pH of the reaction mixture, for example, changingthe pH to be in the range from about 4.0 to about 8.0 units so as tocontrol the ratio of tetrahydrocannabinol to cannabichrome produced bythe above described method.

The present technology also provides a system for producing cannabinoidsor cannabinoid analogs. Such a system comprises a fermentor holding amedium and a plurality of cells, wherein the cells are configured toproduce and secrete cannabinoid synthase, a bioreactor containing areactant, the reactant configured to interact with cannabinoid synthaseto form a first cannabinoid and a second cannabinoid, and a controlmechanism configured to control a condition of the bioreactor. Thelatter control mechanism is used to influence a quantity formed of thefirst cannabinoid relative to a quantity formed of a second cannabinoid.

The system may further comprise a filter configured to at leastpartially separate the plurality of cells from the medium, such thatafter separation the medium containing a cannabinoid synthase isintroduced into a bioreactor. In one embodiment, the enzyme expressed bythe plurality of cells includes a tag that permits the enzyme to bond toa nickel support within the bioreactor. Any Formula I compound orcannabigerolic acid can serve as the substrate for the enzymetetrahydrocannabinoilic acid synthase. Depending on the conditionswithin the bioreactor, tetrahydrocannabinolic acid or cannabichromenicacid is produced as the first cannabinoid and the second cannabinoidwill be the other of tetrahydrocannabinolic acid and cannabichromenicacid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a system for producingcannabinoids or cannabinoid analogs, according to an exemplaryembodiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “acell” includes a plurality of cells, and a reference to “a molecule” isa reference to one or more molecules.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term.

The term “alkyl” refers to a straight or branched chain, saturatedhydrocarbon having the indicated number of carbon atoms. For example,(C₁-C₁₀)alkyl is meant to include but is not limited to methyl, ethyl,propyl, isopropyl, butyl, sec-butyl, tent-butyl, pentyl, isopentyl,neopentyl, hexyl, isohexyl, and neohexyl, etc. An alkyl group can beunsubstituted or optionally substituted with one or more substituents asdescribed herein below.

The term “alkenyl” refers to a straight or branched chain unsaturatedhydrocarbon having the indicated number of carbon atoms and at least onedouble bond. Examples of a (C₂-C₁₀)alkenyl group include, but are notlimited to, ethylene, propylene, 1-butylene, 2-butylene, isobutylene,sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene, 2-hexene,3-hexene, isohexene, 1-heptene, 2-heptene, 3-heptene, isoheptene,1-octene, 2-octene, 3-octene, 4-octene, and isooctene. An alkenyl groupcan be unsubstituted or optionally substituted with one or moresubstituents as described herein below.

The term “alkynyl” refers to a straight or branched chain unsaturatedhydrocarbon having the indicated number of carbon atoms and at least onetriple bond. Examples of a (C₂-C₁₀)alkynyl group include, but are notlimited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne,2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne,3-heptyne, 1-octyne, 2-octyne, 3-octyne and 4-octyne. An alkynyl groupcan be unsubstituted or optionally substituted with one or moresubstituents as described herein below.

The term “alkoxy” refers to an —O-alkyl group having the indicatednumber of carbon atoms. For example, a (C₁-C₆)alkoxy group includes—O-methyl, —O—ethyl, —O—propyl, —O—isopropyl, —O—butyl, —O-sec-butyl,—O-tert-butyl, —O-pentyl, —O-isopentyl, —O-neopentyl, —O-hexyl,—O-isohexyl, and —O-neohexyl.

The term “aryl” refers to a 3- to 14-member monocyclic, bicyclic,tricyclic, or polycyclic aromatic hydrocarbon ring system. Examples ofan aryl group include naphthyl, pyrenyl, and anthracyl. An aryl groupcan be unsubstituted or optionally substituted with one or moresubstituents as described herein below.

The terms “alkylene,” “alkenylene,” and “arylene,” alone or as part ofanother sub stituent, means a divalent radical derived from an alkyl,cycloalkyl, alkenyl, aryl, or heteroaryl group, respectively, asexemplified by —CH₂CH₂CH₂CH₂—. For alkylene, alkenyl, or aryl linkinggroups, no orientation of the linking group is implied.

The term “halogen” and “halo” refers to —F, —Cl, —Br or —I.

The term “heteroatom” is meant to include oxygen (O), nitrogen (N), andsulfur (S).

A “hydroxyl” or “hydroxy” refers to an —OH group.

The term “hydroxyalkyl,” refers to an alkyl group having the indicatednumber of carbon atoms wherein one or more of the alkyl group's hydrogenatoms is replaced with an —OH group. Examples of hydroxyalkyl groupsinclude, but are not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂CH₂OH, and branchedversions thereof.

The term “cycloalkyl” refer to monocyclic, bicyclic, tricyclic, orpolycyclic, 3- to 14-membered ring systems, which are either saturated,unsaturated or aromatic. The heterocycle may be attached via anyheteroatom or carbon atom. Cycloalkyl include aryls and hetroaryls asdefined above. Representative examples of cycloalky include, but are notlimited to, cycloethyl, cyclopropyl, cycloisopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclopropene, cyclobutene, cyclopentene,cyclohexene, phenyl, naphthyl, anthracyl, benzofuranyl, andbenzothiophenyl. A cycloalkyl group can be unsubstituted or optionallysubstituted with one or more substituents as described herein below.

The term ‘nitrile or cyano” can be used interchangeably and refer to a—CN group which is bound to a carbon atom of a heteroaryl ring, arylring and a heterocycloalkyl ring.

The term “amine or amino” refers to an —NR_(c)R_(d) group wherein R_(c)and R_(d) each independently refer to a hydrogen, (C₁-C₈)alkyl, aryl,heteroaryl, heterocycloalkyl, (C₁-C₈)haloalkyl, and (C₁-C₆)hydroxyalkylgroup.

The term “alkylaryl” refers to C₁-C₈ alkyl group in which at least onehydrogen atom of the C₁-C₈ alkyl chain is replaced by an aryl atom,which may be optionally substituted with one or more substituents asdescribed herein below. Examples of alkylaryl groups include, but arenot limited to, methylphenyl, ethylnaphthyl, propylphenyl, andbutylphenyl groups.

“Arylalkylene” refers to a divalent alkylene wherein one or morehydrogen atoms in the C₁-C₁₀ alkylene group is replaced by a(C₃-C₁₄)aryl group. Examples of (C₃-C₁₄)aryl-(C₁-C₁₀)alkylene groupsinclude without limitation 1-phenylbutylene, phenyl-2-butylene,1-phenyl-2-methylpropylene, phenylmethylene, phenylpropylene, andnaphthylethylene.

“Arylalkenylene” refers to a divalent alkenylene wherein one or morehydrogen atoms in the C₂-C₁₀ alkenylene group is replaced by a(C₃-C₁₄)aryl group.

The term “arylalkynylene” refers to a divalent alkynylene wherein one ormore hydrogen atoms in the C₂-C₁₀ alkynylene group is replaced by a(C₃-C₁₄)aryl group.

The terms “carboxyl” and “carboxylate” include such moieties as may berepresented by the general formulas:

E in the formula is a bond or O and R^(f) individually is H, alkyl,alkenyl, aryl, or a pharmaceutically acceptable salt. Where E is O, andR^(f) is as defined above, the moiety is referred to herein as acarboxyl group, and particularly when R^(f) is a hydrogen, the formularepresents a “carboxylic acid”. In general, where the expressly shownoxygen is replaced by sulfur, the formula represents a “thiocarbonyl”group.

Unless otherwise indicated, “stereoisomer” means one stereoisomer of acompound that is substantially free of other stereoisomers of thatcompound. Thus, a stereomerically pure compound having one chiral centerwill be substantially free of the opposite enantiomer of the compound. Astereomerically pure compound having two chiral centers will besubstantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, for example greater than about 90%by weight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

If there is a discrepancy between a depicted structure and a name giventhat structure, then the depicted structure controls. Additionally, ifthe stereochemistry of a structure or a portion of a structure is notindicated with, for example, bold or dashed lines, the structure orportion of the structure is to be interpreted as encompassing allstereoisomers of it.

The present invention focuses on methodologies for enzyme catalyzedsynthesis of cannabinoids or cannabinoid analogs in a cell-freeenvironment. Also described is an apparatus for the ex vivo manufactureof cannabinoids and analogs of cannabinoids. The term “analog” refers toa compound that is structurally related to naturally occurringcannabinoids, but whose chemical and biological properties may differfrom naturally occurring cannabinoids. In the present context, analog oranalogs refer compounds that may not exhibit one or more unwanted sideeffects of a naturally occurring cannabinoid. Analog also refers to acompound that is derived from a naturally occurring cannabinoid bychemical, biological or a semi-synthetic transformation of the naturallyoccurring cannabinoid.

Illustrative of cannabinoid compounds included in the inventions andwithout limitation are cannabinol, cannabidiol, Δ9-tetrahydrocannabinol,Δ8-tetrahydrocannabinol, 11-hydroxy-tetrahydrocannabinol,11-hydroxy-Δ9-tetrahydrocannabinol, levonantradol,Δ11-tetrahydrocannabinol, tetrahydrocannabivarin, dronabinol,arnandamide and nabilone, as well as natural or synthetic molecules thathave a basic cannabinoid structure and are modified synthetically toprovide a cannabinoid analog.

The present technology also relates to the large scale cloning andexpression of the enzymes that play a role in the biosynthesis ofcannabinoids and the use of an eukaryotic expression system for themanufacture of cannabinoids and cannabinoid analogs. Exemplary ofeukaryotic cells suitable for cloning and expression of the cannabinoidsynthase enzymes include without limitation E coli, yeast andbaculovirus hosts. In an embodiment of this technology, is disclosed amethod for the large-scale production of several cannabinoid synthaseenzymes including tetrahydrocannabinolic acid synthase (THCA synthase),cannabichromenic acid synthase (CBCA synthase) and cannabidiolic acidsynthase (CBDA synthase) using the pink Pichia yeast expression system.Accordingly, the large scale production of these enzymes can be carriedout by transforming yeast with a DNA construct that comprises a gene forTHCA synthase, CBCA synthase, or CBDA synthase, and culturing thetransformed yeast cells under conditions suitable for promoting theexpression of a functionally active enzyme.

The sequences for the genes for THCA synthase and CBDA synthase wereobtained from a publicly available database and their protein codingregions were altered to optimize protein expression in Pichia Pastorisyeast cells. Codon optimization was carried out using the GeneArt®program from Invitrogen.

Additionally, both genes were modified to include the yeast alphasecretory sequence as well as codons for the inclusion of a His-tag inthe expressed protein. The former was inserted at the 5′-end of the geneand is necessary for the extracellular secretion of the expressedprotein. Codons for the His-tag are present at the 3′-end of the geneand are introduced to facilitate the purification of the expressedprotein by affinity chromatography.

These chimeric sequences, alpha-CBDA synthase and alpha-THCA synthase,were inserted into pPink-HC vector (Invitrogen®), and were used toobtain Top 10 F⁻ transformed E. coli cells which were stored as agarstabs for future use. Prior to transformation of yeast cells, the vectorcontaining the cannabinoid synthase gene of interest (GOI) was isolatedfrom agar stabs containing transformed E. coli cells, and linearizedusing PmeI or SpeI restriction enzymes. The linearized plasmids wereelectroporated into Pichia pastoris pepB deficient mutant cells usingPichiaPink™ Yeast Expression Systems (Invitrogen®). Linearization usingthe restriction enzyme PmeI directs the insert into the AOX1 promoterregion of the pichia genome, while the restriction enzyme SpeI directsthe insert into the TRP gene.

The transformed yeast cells were grown on adenine-deficient selectiveplates and a color-based screen was used to identify positivetransformants. Pursuant to this screening methodology, red/pink coloniessignal transformants that do not carry the gene of interest as well astransformants carrying a limited number of copies of the gene ofinterest integrated into the genome of Pichia pastoris. White colonieson the other hand , indicate transformants having multiple copies of thegene of interest. Typically, cells having 6-10 copies of the gene ofinterest are desired for obtaining large amounts of recombinant protein,for example, about 1.0 g to about 2.0 g of protein per liter of culture.An enzyme assay that quantitated the conversion of substratecannabigerolic acid (CBGA) to THCA or CBDA products upon induction ofTHCA synthase or CBDA synthase by the addition of methanol totransformed yeast cells was used to identify white colonies of yeastcells that produced greater than 20% conversion of substrate to product.

Accordingly, individual white colonies of yeast cells carrying the THCAsynthase gene or the CBDA synthase gene were separately cultured inflasks using BMGY medium, followed by induction by growth in BMMYmedium, to induce the expression of THCA synthase or CBDA synthase asfurther described below. Briefly, the medium containing the enzyme ineach culture was separated from the cells and a known amount of thesubstrate CBGA was added to the medium from each culture flask.Following incubation, each culture flask was analyzed to quantitate thepercent conversion of CBGA to product. Cultures of transformants showinggreater than 20% conversion will be used for the commercial synthesis ofcannabinoids or cannabinoid analogs pursuant to methodologies of theinvention.

The cannabinoid synthase enzymes, THCA synthase and CBDA synthase,obtained using the PichiaPink™ Yeast Expression system described above,can be used for the manufacture of cannabinoids or an analog of acannabinoid. In one embodiment is provided a method for producing acannabinoid or a cannabinoid analog by selecting a Formula I compoundand a cannabinoid synthase as a catalyst for transforming the Formula Icompound to a cannabinoid or a cannabinoid analog.

In Formula I, R can be selected from hydroxyl (—OH), halogen, thiol(—SH), or a —NR_(a)R_(b) group. Substituent groups R₁ and R₂ are eachindependently selected from the group consisting of —H, —C(O)R_(a),—OR_(a), an optionally substituted C₁-C₁₀ linear or branched alkylene,an optionally substituted C₂-C₁₀ linear or branched alkenylene, anoptionally substituted C₂-C₁₀ linear or branched alkynylene, anoptionally substituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene.Alternatively, R₁ and R₂ together with the carbon atoms to which theyare bonded form a C₅-C₁₀ cyclic ring. For compounds according to FormulaI, R₃ is selected from the group consisting of H, —C(O)R_(a) and C₁-C₁₀linear or branched alkyl and R_(a) and R_(b) are each independently —H,—OH, (C₁-C₁₀) linear or branched alkyl, —SH, 13 NH₂, or a C₃-C₁₀cycloalkyl.

The cannabinoid or cannabinoid analog obtained by contacting a Formula Icompound with a cannabinoid synthase can be isolated, purified and usedas a therapeutic or and the cannabinoid or cannabionoid analog canundergo an optional decarboxylation step to convert, for example,cannabichromenic acid (CBCA) to cannabichromene (CBC) prior to thelatter's use as a pharmaceutical agent or a nutraceutical agent.

For certain embodiments the cannabinoid synthase is cannabidiolic acidsynthase. For other aspects of this technology, the cannabinoid synthaseis tetrahydrocannabinolic acid synthase or a cannabichromene acidsynthase. For certain Formula I compounds R₁ and R₂ together with thering carbon atoms to which they are bonded form a C₅-C₁₀ cyclic ring.For such Formula I compounds one or more carbon atoms of the ring can besubstituted with a heteroatom selected from oxygen, sulfur or nitrogen.

For compounds according to Formula I, R₂ can be a linear alkylene or abranched alkylene. Exemplary of linear alkylenes include withoutlimitation CH₃, C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅ and C₈H₁₇.Illustrative of branched alkylenes are groups selected from, iso-propyl,sec-butyl, iso-butyl, neopentyl, 2-methyl hexyl, or 2,3-dimethyl hexylgroups. In some embodiments, R₂ can be an optionally substituted linearor branched alkylene in which one or more hydrogen atoms is replacedwithout limitation with a group selected from chlorine, fluorine,bromine, nitro, amino, hydroxyl, phenyl, or benzyl group.

For certain Formula I compounds R₂ is a C₂-C₁₀ alkenylene and isselected from the group consisting of

with R₄ being a linear or branched alkylene as described above. When R₂is a C₂-C₁₀ linear or branched alkynylene, R₂ can be

Alternatively, R₂ in Formula I is

substituent X is a group selected from —OH, —SH, or NR_(a)R_(b) andgroups R_(a) and R_(b). are as defined above.

Many of the naturally occurring cannabinoids are produced as theircarboxylic acid derivatives in plants. Their psychostimulatory activityis enhanced, however, following decarboxylation which occurs upon theapplication of heat to the cannabinoid acid containing plant tissue orby drying the plant material prior to use. Cannabinoid and cannabinoidanalogs synthesized using the inventive method can also have acarboxylic acid (—COOH) group as the R₁ substituent and such compoundsmay undergo an optional decarboxylation step prior to their use aspharmaceutical or nutraceutical agents. Exemplary of such a cannabinoidor cannabinoid analog is the compound obtained by contacting a Formula Ispecies in which R is —OH, R₁ is —COOH, R₂ is C₅H₁₁ and R₃ is —H with acannabinoid synthase.

The synthesis, isolation and purification of cannabinoids or cannabinoidanalogs can be improved by immobilization of a cannabinoid synthase to asolid support, or by encapsulation of the synthase within a liposome. Inone aspect of the synthesis, the enzyme is immobilized to a solidsupport. Such immobilization is advantageous, since it permits recyclingand reuse of the immobilized enzyme which significantly reduces thecosts associated with the manufacture of pharmaceutical gradecannabinoids or cannabinoid analogs. Immobilization of the enzyme alsopermits ease of use and recovery of the enzyme catalyst, ease ofpurification of the desired product, preservation of the enantiomericexcess (ee) of the final product and an overall improvement in the yieldof the product. Typically, the enantiomeric purity of a cannabinoid or acannabinoid analog according to the claimed method is from about 90% eeto about 100% ee, for instance, a cannabinoid or a cannabinoid analogproduced using the inventive methodology can have an enantiomeric purityof about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee,about 96% ee, about 97% ee, about 98% ee and about 99% ee.

Typically, the enzyme to be immobilized can be absorbed onto a solidsupport, adsorbed onto a support, covalently linked to a support or canbe immobilized onto a solid support through ionic interactions. In oneembodiment, the cannabinoid synthase is covalently linked to a solidsupport. Suitable strategies for linking an enzyme to a solid supportare well known in the biochemical art and include covalent linkagesbetween an appropriately functionalized support and a side chain of anamino acid group or through covalent linkages using appropriatelyfunctionalized linkers or spacers to separate the support from theenzyme. The term “linker” refers to any group that separates the supportfrom the enzyme. Accordingly, a linker is a group that is covalentlytethered at one end to a group on the surface of the support and isattached to the enzyme at the other end. Illustrative linkers include(C₁-C₁₀)alkylene linker polymers of ethylene glycol such as a—(OCH₂—CH₂)_(n)—O— group, where n is an integer from 0 to 10,—(C₁-C₁₀)alkylene-NH—, —(C₁-C₁₀)alkylenesiloxy, or a—(C₁-C₁₀)alkylene-C(O)—.

Supports suitable for immobilizing enzymes include without limitationAmeberlite resins, Duolite resins, acrylic resins such as Eupergit® C,DEAE-Sephadex and gels made using polyvinyl alcohol can be used assupports for immobilizing the cannabinoid synthase enzymes of thepresent technology.

Cannabinoids exert different physiological properties and are known tolessen pain, stimulate appetite and have been tested as candidatetherapeutics for treating a variety of disease conditions such asallergies, inflammation, infection, epilepsy, depression, migraine,bipolar disorders, anxiety disorder, and glaucoma. The physiologicaleffect exerted by a cannabinoid depends in large part to its ability tostimulate or deactivate the cannabinoid receptors, for instance the CB1,CB2 and CB3 receptors. Since modulation of receptor activity depends onthe binding interactions and the orientation of a ligand within thecannabinoid receptors active site, it follows that the nature andorientation of substituent groups attached to a cannabinoid or acannabinoid analog will affect the pharmaceutical properties exhibitedby such compounds.

In one embodiment is provided a method for the manufacture cannabinoidshaving structurally distinct and diverse substituent groups attached toa central core. Such compounds are expected to exhibit differentpharmaceutically beneficial properties. Structural diversity will beintroduced by contacting an appropriately substituted Formula IIIcompound with a Formula IV compound in the presence of an enzyme, suchas GPP olivetolate geranyltransferase (a polyketide synthase), to give aFormula II compound. Scheme 1 structurally illustrates the protocol forsynthesizing a Formula II compound pursuant to this embodiment.

Depending on the nature and type of substituent groups at R, R₁, R₂, R₃and R₅, in Formulae III and IV, the inventive method permits thesynthesis of Formula II compounds having different substituent groupssurrounding the central phenyl core as precursors or synthons for themanufacture of a cannabinoid or a cannabinoid analog. According to thismethod, therefore, the cannabinoid or cannabinoid analogs can beobtained by contacting a Formula II compound with a cannabinoidsynthase, for example, THCA synthase, CBCA synthase or a CBDA synthase,followed by isolation and decarboxylation of the obtained product togive a cannabinoid or a cannabinoid analog.

In Formula III, R can be selected from hydroxyl (—OH), halogen, thiol(—SH), or a —NR_(a)R_(b) group. Substituents R₁ and R₂ are eachindependently selected from the group consisting of —H, —C(O)R_(a),—OR_(a), an optionally substituted linear or branched (C₁-C₁₀)alkylene,an optionally substituted linear or branched (C₂-C₁₀)alkenylene, anoptionally substituted linear or branched (C₂-C₁₀)alkynylene, anoptionally substituted C₃-C₁₀ aryl, an optionally substituted C₃-C₁₀cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene.

In certain embodiments R₁ and R₂ together with the carbon atoms to whichthey are bonded form a C₅-C₁₀ cyclic ring and R₃ is selected from thegroup consisting of H, —C(O)R_(a) and C₁-C₁₀ linear or branched alkyl.

R₅ in Formula IV can be a linear or branched (C₁-C₁₀)alkylene, a linearor branched (C₂-C₁₀)alkenylene, a linear or branched (C₂-C₁₀)alkynylene,—C(O)—(C₁-C₁₀)alkylene, —C(O)—(C₂-C₁₀)alkenylene and—C(O)—(C₂-C₁₀)alkynylene. For Formulae II, III and IV compounds anyalkylene, alkenylene, alkynylene, aryl, arylalkylene, or cycloalkylgroup can be further substituted with one or more groups selected fromthe group consisting of —OH, halogen, —NR_(b)R_(c), —C(O)R_(a),—C(O)NR_(b)R_(c), (C₁-C₁₀)alkyl, —CN, (C₁-C₄)alkoxy, (C₁-C₄)haloalkyl,and (C-C₄)hydroxyalkyl with R_(a), R_(b) and R_(c) each independentlybeing selected from —H, —OH, or (C₁-C₁₀) linear or branched alkyl, —SH,—NH₂, or a C₃-C₁₀ cycloalkyl.

According to one embodiment, R₅ in Formula IV can be a(C₂-C₁₀)alkenylene selected from

with R₄ being a linear alkylene selected from the group consisting ofCH₃, C₂H₅, C₃H₇, C₄H₉, C₅H₁₁,

C₆H₁₃, C₇H₁₅ and C₈H₁₇. For certain Formula IV compounds R₅ is

and group R₆ is selected from (C₁-C₁₀)alkylene, (C₂-C₁₀)alkenylene, —OH,—SH, NO₂, F, Cl, Br, —NH₂,or a —NHR_(a) where R_(a) is as defined above.

Enzymes are very specific with respect to the type of chemical reactionsthey catalyze and the nature and type of substrates that are involved inthese reactions. Enzymes also exhibit a high level of stereospecificity,regiospecificity and chemoselectivity. It was therefore unexpected, whenthe present inventors observed that the enzyme THCA synthase couldproduce two different products, tetrahydrocannabinolic acid (THCA) andcannabichromenic acid (CBCA) using the same substrate cannabigerolicacid (CBGA), depending on the conditions under which the cyclizationreaction is catalyzed.

Accordingly, the effects of temperature, pH, solvent, ionic strength andincubation times on the distribution ratio of THCA to CBCA products werestudied. In one embodiment, the effect of solvent on productdistribution ratio was evaluated. Cannabinoids are lipohilic in natureand are poorly solubilized in aqueous solvents. The poor solubility ofcannabinoids in aqueous solvent has prevented the development of ex-vivoenzyme catalyzed methodologies for the synthesis of cannabinoids andcannabinoid analogs. The present inventors surprisingly found that THCAsynthase retained its catalytic activity in a solvent mixture containingbuffer and a non-aqueous solvent, such as dimethyl sulfoxide (DMSO),dimethyl formamide (DMF), iso-propoyl alcohol and cyclodextrin if theconcentration of non-aqueous solvent in the mixture was maintained below40% v/v.

For instance, enzyme catalysis was most effective when the concentrationof the non-aqueous solvent was about 20%. The catalytic rate decreasesslightly as concentration of the non-aqueous solvent is increased. Thus,20% DMSO gave the highest catalytic rate, approximately 2.5-fold fasterthan catalysis in buffer alone. Table 1 illustrates the effect ofconcentration of non-aqueous solvent on the rate of catalytic activity.

As further illustrated in this table, the concentration of non-aqueoussolvent can alter the ratio of THCA to CBCA produced as product. Thus,greater levels of THCA are obtained when the concentration of DMSO inbuffer is less that 20%, with a THCA:CBCA distribution ratio of 10:1observed when 10% DMSO is used as the solvent.

TABLE 1 DMSO FASTER THCA:CBCA 0% 1X 10% 1.2X 10:1  20% 2.5X 5:1 25% —1:1 30% 0.3X

It was observed that the addition of a detergent such as sodium dodecylsulfate (SDS), to a 100 mM sodium phosphate buffer did not impact thepercent conversion of substrate CBGA to product. In contrast, additionof SDS to 100 mM citrate buffer containing cyclodextrin destroyed THCAsynthases ability to catalyze reaction. Only 8% of the substrate wasconverted to product under these reaction conditions with the formationof THCA being favored as product under these reaction conditions.

Cannabigerolic acid (CBGA) serves as a common substrate for differentcannabinoid synthase enzymes during the biosynthesis of naturallyoccurring cannabinoids. For instance, previous studies have suggestedthat the enzymes THCA synthase, CBDA synthase and CBCA synthase to eachuse CBGA as their substrate during the biosynthesis oftetrahydrocannabinolic acid, cannabidiolic acid and cannabichromenicacid respectively.

Thus, during a study aimed at evaluating the optimal pH for THCAsynthase activity, the present inventors were surprised to note thatdepending on the pH at which catalysis was carried out the enzyme THCAsynthase catalyzed the conversion of substrate CBGA to either THCA orCBCA. This observation was unexpected since a single enzyme THCAsynthase, could be responsible for the formation of two differentcannabinoid products in plants.

Table 3 illustrates the results of a pH study that was aimed atidentifying the effect of pH on product ratio, namely, the ratio of THCAto CBCA compounds produced when THCA synthase is contacted with CBGA assubstrate. The pH of the reaction mixture in this study ranged from a pHof 4.0 to 8.0. Higher pH values were not tested since THCA synthase iscatalytically inactive at a pH greater than 8.0.

As illustrated in Table 3, THCA is preferentially synthesized at pHvalues less than 6.0. In fact, THCA is the major product when catalysisis carried out at a pH of 4.0. Increasing the pH of the reaction mixturealtered the product distribution ratio with approximately 30% CBCA beingproduced as product at a pH of 5.0 and around 15% CBCA being produced asproduct at a pH value of 6.0. A further increase in pH of the reactionmixture, such as an increase of the pH of the reaction mixture to a pHof 7.0 gave CBCA exclusively as the product of THCA synthase catalyzedconversion of CBGA. Any further increases in pH, moreover, reduced oreliminated catalytic activity of THCA synthase, with no product observedfor a reaction mixture at a pH of 8.0.

TABLE 3 pH THCA CBCA 4 100 Trace 5 70 30 6 85 15 7 0 100 8 0 0

Accordingly, in one embodiment the invention provides a method forproducing a tetrahydrocannabinol, cannabichrome or bothtetrahydrocannabinol and cannabichrome, or their analogs by selecting aFormula V compound as a reactant, contacting the Formula V compound withtetrahydrocannabinolic acid synthase (THCA synthase) and modifying atleast one property of the reaction mixture to obtaintetrahydrocannabinol, cannabichrome or both tetrahydrocannabinol andcannabichrome, or their analogs as products.

In Formula V, R is selected from —OH, halogen, —SH, or a —NR_(a)R_(b)group. Substituents R₁ and R₂ are each independently selected from thegroup consisting of —H, —C(O)R_(a), —OR_(a), an optionally substitutedC₁-C₁₀ linear or branched alkylene, an optionally substituted C₂-C₁₀linear or branched alkenylene, an optionally substituted C₂-C₁₀ linearor branched alkynylene, an optionally substituted C₃-C₁₀ aryl, anoptionally substituted C₃-C₁₀ cycloalkyl, (C₃-C₁₀)aryl-(C₁-C₁₀)alkylene,(C₃-C₁₀)aryl-(C₂-C₁₀)alkenylene, and (C₃-C₁₀)aryl-(C₁-C₁₀)alkynylene.

For certain embodiments, R₁ and R₂ together with the ring carbon atomsto which they are bonded form a C₅-C₁₀ cyclic ring in which one or morecarbon atoms can optionally be replaced with one or more heteroatomsselected from oxygen, sulfur, nitrogen or a —NR^(a) group. R₃ in FormulaV, can be a group selected from H, —C(O)R_(a) or a (C₁-C₁₀) linear orbranched alkyl and substituents R_(a) and R_(b) are each independentlyselected from —H, —OH, —SH, NH₂, (C₁-C₁₀) linear or branched alkyl, or aC₃-C₁₀ cycloalkyl moiety.

Any physical property known to have an effect on enzyme activity andcatalysis can be modulated to alter the ratio of the products THCA toCBCA. In one embodiment, therefore, the pH of the reaction mixture waschanged to modulate the ratio of THCA to CBCA produced enzymatically asproducts. Pursuant to this synthetic methodology, catalysis at a lowerpH in the range from about 4.0 to about 6.0 favored the formation ofTHCA as product while catalysis at a neural pH in the range from about6.5 to about 7.5 favored the formation of CBCA as product.

Thus, the present inventors have shown that it is possible to controlthe formation of THCA or CBCA as the product of catalysis by controllingthe pH of the reaction mixture.

Other physical properties such as the compositional make-up of thereaction solvent, ionic strength, temperature, pressure, viscosity ofthe reaction medium and concentration of reagents can also alter productratio. Many of these physical parameters, in fact, play an importantrole in modulating catalysis during the large scale manufacture ofcannabinoids or cannabinoid analogs using a bioreactor.

Thus, in one embodiment is provided a system (100) for producing acannabinoid or a cannabinoid analog by controlling a condition thatinfluences the quantity of a first cannabinoid or its analog formed inrelation to the quantity of a second cannabinoid or its analog. Thesystem (100), shown schematically in FIG. 1, may comprise a fermentor(2), a filter (18), a bioreactor (10), and a control mechanism (20). Adescription of the System as represented in FIG. 1 is provided below.

The fermentor (2) holds cell culture medium (4) and a plurality of cells(6). The cells (6) are configured to produce and secrete a cannabinoidsynthase (8). The cells (6) used in the fermentor (2) for themanufacture of a cannabinoid synthase (8) can be any eukaryotic cellthat has been genetically modified to include a nucleic acid sequence ora gene that encodes a cannabinoid synthase protein. In certainembodiments, the nucleic acid sequence that encodes a cannabinoidsynthase protein is modified to include an yeast alpha secretionsequence at its 5′ end and to incorporate a 6-residue histidine tag atits 3′ end. The addition of the yeast alpha secretion sequence permitssecretion of the cannabinoid synthase protein into the medium used foreukaryotic cell growth. The extracellular secretion of the cannabinoidsynthase (8) is advantageous, since it facilitates the separation andtransport of the enzyme between the fermentor (2) and the bioreactor(10) using the filter (18). Following production of cannabinoid synthase(8) in the fermentor (2), the supernatant (e.g. medium (4), cells (6),and cannabinoid synthase (8) is transported along path (24) to thefilter (18). Path (24) may be a pipe or any other pathway suitable fortransporting the supernatant.

The filter (18) may filter the supernatant to at least partiallyseparate the cells (6) from the medium (4) containing the expressedenzyme. Typically, the filter (18) separates at least 80% of the totalcells (6) from the medium (4). For certain embodiments, the filter (18)separates at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% of the total cells (6) from the medium(4) prior to the introduction of this medium (4) into the bioreactor(10). Following filtration, the cells (6) are transported back to thefermentor (2) along path (26). In one embodiment, the filter (18) can bea filtration and purification system that includes multiple filters andreservoirs to purify the cannabinoid synthase (8).

After passing through the filter (18), the cannabinoid synthase (8)flows into the bioreactor (10) along path (28) and enters the bioreactor(10) through inlet (30). The bioreactor (10) also includes an inlet (32)for reactants, such as the substrate CBGA or other substrates accordingto the Formula I compound described above. Thus, as shown in FIG. 1, thebioreactor (10) contains a reactant (12) that is configured to interactwith cannabinoid synthase (8) to form a first cannabinoid (14). Thebioreactor (10) may also provide the environment for synthesis of asecond cannabinoid (16). The second cannabinoid (16) may be producedusing the same type of cannabinoid synthase (8) and a substrate as thefirst cannabinoid (14). For example, both the first cannabinoid (14) andthe second cannabinoid (16) may be produced using CBGA as reactant (12)and THCA synthase as the cannabinoid synthase (8). In this embodiment,the first cannabinoid (14) may be THCA and the second cannabinoid (16)may be CBCA. In an alternative embodiment, the second cannabinoid (16)may be synthesized using a substrate or cannabinoid synthase differentfrom those used in synthesis of the first cannabinoid (14).

The bioreactor (10) can be a column bioreactor having a solid supportthat is impregnated with divalent metal ions or a support whose surfaceis functionalized with divalent metal ions. Typically, sepharose,agarose or other biopolymers are used as supports for binding divalentmetal ions such as nickel, cobalt, magnesium and manganese. Suchsupports have a strong affinity for the histidine tag that is present onthe expressed cannabinoid synthase (8) and can be used to sequester thesynthase and separate it from other non-essential proteins and debristhat may interfere or impede cannabinoid synthesis.

The bioreactor (10) used for synthesizing cannabinoids is configured forbatch and continuous synthetic processes to permit commercial productionof pharmaceutically useful cannabinoids. In one embodiment, thebioreactor (10) is configured for batch synthesis in which thecomposition of the medium (4), concentration of the enzyme and substrateare fixed at the beginning of the process and not allowed to changeduring catalysis. Synthesis is terminated when the concentration of thedesired product in the medium of the bioreactor (10) reaches apredetermined value or the concentration of substrate falls below apredetermined level, such as to a level where there is no detectablecatalytic conversion of substrate to product.

In one embodiment, therefore, the His-tagged cannabinoid synthase (8) issequestered onto a nickel containing resin support within the bioreactorcolumn prior to the introduction of a known amount of substrate, forexample, cannabigerolic acid (CBGA), or a Formulae I, II or V compoundinto the bioreactor (10). In an alternate embodiment, cannabigerolicacid (CBGA), or a Formulae I, II or V compound can be present within thebioreactor (10) having a nickel resin support prior to the introductionof the medium (4) containing a cannabinoid synthase (8) into thebioreactor (10). In either case, a known amount of the enzyme iscontacted with a known amount of a Formulae I, II or V compound or CBGAas substrate to synthesize a cannabinoid or a cannabinoid analog asproduct, such as the first cannabinoid (14) or the second cannabinoid(16).

The progress of the reaction within the bioreactor (10) can be monitoredperiodically or continuously. For instance, an optical monitoring systemmay be utilized to detect the concentration of product in the medium (4)within the bioreactor (10) as a function of time. Alternatively, thedecrease in the concentration of substrate can be monitored to signaltermination of synthesis. The cannabinoid product thus produced can bereadily recovered from the medium using standard solvent extraction orchromatographic purification methods. The monitoring system may be partof or may interact with the control mechanism (20), described furtherbelow.

An alternative to the batch process mode is the continuous process modein which a defined amount of substrate and medium (4) are continuouslyadded to the bioreactor (10) while an equal amount of medium (4)containing the cannabinoid product is simultaneously removed from thebioreactor (10) to maintain a constant rate for formation of product.Medium (4) can enter the bioreactor (10) through inlet (32) and exit thebioreactor 10 through outlet (34). Methods of modulating theconcentration of substrate, enzyme and other factors implicated tomaximize the rate of product formation are known in the art.

The conditions of the bioreactor (10) can be controlled using a controlmechanism (20). The control mechanism (20) may be coupled to thebioreactor (10) or, alternatively, may interact with the bioreactor (10)wirelessly or remotely. The control mechanism (20) can control at leastone condition of the bioreactor (10) so as to influence a quantityformed of the first cannabinoid (14) relative to a quantity formed of asecond cannabinoid (16). For example, in one embodiment, the cannabinoidsynthase (8) is THCA synthase and is produced by genetically engineeredPichia pastoris yeast cells. As described above, contact of this enzymewith cannabigerolic acid permits the production of both THCA or CBCA.One condition that influences the quantity of THCA produced relative toCBCA (e.g. the ratio of THCA to CBCA) is the pH of the medium (4) in thebioreactor (10). Other conditions within the bioreactor 10 may alsoinfluence the relative quantities of a first cannabinoid (14) (e.g.THCA) and second cannabinoid (16) (e.g. CBCA) produced in the bioreactor(10), such as temperature, pressure, and flow rate. In one embodiment, achange in condition (e.g. pH, temperature, pressure, and/or flow rate)can cause a shift from formation of the first cannabinoid (14) ingreater quantities relative to the second cannabinoid (16) to formationof the second cannabinoid (16) in greater quantities relative to thefirst cannabinoid (14).

In another embodiment, the control mechanism (20) can also be used tocontrol the conditions of the fermentor (2), such the oxygen level,agitation, pH, and feed rate. The control mechanism (20) may alsocontrol the flow of materials (e.g. by controlling pumps) into and outof the fermentor (2), filter (18), and bioreactor (10).

The control mechanism (20) can include a processing circuit having aprocessor and memory device. The processor can be implemented as ageneral purpose processor, an application specific integrated circuit(ASIC), one or more field programmable gate arrays (FPGAs), a group ofprocessing components, or other suitable electronic processingcomponents. The memory device (e.g., memory, memory unit, storagedevice, etc.) is one or more devices (e.g., RAM, ROM, Flash memory, harddisk storage, etc.) for storing data and/or computer code for completingor facilitating the various processes and functions described in thepresent application, such as controlling the pH, temperature, andpressure of the bioreactor (10), or altering the flow rate of medium (4)into or out of the bioreactor (10). The memory device may be or includevolatile memory or non-volatile memory. The memory device may includedatabase components, object code components, script components, or anyother type of information structure for supporting the variousactivities and information structures described in the presentapplication. According to one embodiment, the memory device iscommunicably connected to the processor via the processing circuit andincludes computer code for executing (e.g., by the processing circuitand/or processor) one or more processes described herein.

The present disclosure contemplates methods, systems and programproducts on any machine-readable media for accomplishing variousoperations, such as controlling the conditions of the bioreactor 10. Theembodiments of the present disclosure may be implemented using existingcomputer processors, or by a special purpose computer processor for anappropriate system, incorporated for this or another purpose, or by ahardwired system. Embodiments within the scope of the present disclosureinclude program products comprising machine-readable media for carryingor having machine-executable instructions or data structures storedthereon. Such machine-readable media can be any available media that canbe accessed by a general purpose or special purpose computer or othermachine with a processor. By way of example, such machine-readable mediacan comprise RAM, ROM, EPROM, EEPROM, CD-ROM or other optical diskstorage, magnetic disk storage, other magnetic storage devices, solidstate storage devices, or any other medium which can be used to carry orstore desired program code in the form of machine-executableinstructions or data structures and which can be accessed by a generalpurpose or special purpose computer or other machine with a processor.When information is transferred or provided over a network or anothercommunications connection (either hardwired, wireless, or a combinationof hardwired or wireless) to a machine, the machine properly views theconnection as a machine-readable medium. Thus, any such connection isproperly termed a machine-readable medium. Combinations of the above arealso included within the scope of machine-readable media.Machine-executable instructions include, for example, instructions anddata which cause a general purpose computer, special purpose computer,or special purpose processing machines to perform a certain function orgroup of functions.

The control mechanism (20) may further include additional devices, suchas a keyboard (22) and display (36), to allow a user to interact withthe control mechanism (20) to control the conditions of bioreactor 10.For example, the display may include a screen to allow a user to monitorchanges in pH, temperature, pressure, and flow rate of the bioreactor(10), or to monitor any other condition of the system for producingcannabinoids or cannabinoid analogs.

The construction and arrangement of the system for producingcannabinoids or cannabinoid analogs as shown in the various exemplaryembodiments are illustrative only. Although only a few embodiments havebeen described in detail in this disclosure, many modifications arepossible (e.g., variations in sizes, dimensions, structures, shapes andproportions of the various elements, values of parameters, use ofmaterials, colors, orientations, etc.). For example, the position ofelements may be reversed or otherwise varied and the nature or number ofdiscrete elements or positions may be altered or varied. Accordingly,all such modifications are intended to be included within the scope ofthe present disclosure. Furthermore, the order or sequence of anyprocess or method steps may be varied or re-sequenced according toalternative embodiments. Other substitutions, modifications, changes,and omissions may be made in the design, operating conditions andarrangement of the exemplary embodiments without departing from thescope of the present disclosure.

The present technology is further described by the following exampleswhich are not meant to limit the scope of the claims.

EXAMPLES

A. Molecular Cloning, Screening and Expression of Protein from HighYield Yeast Transformants

1. Restriction Digestion.

THCA α plasmid DNA or CBDA α plasmid DNA were linearized by digestingthe plasmid with Pme I or Spe I restriction enzymes at 37° C. for theappropriate time. The DNA was then ethanol precipitated using a 1:10diluted solution of 3M sodium acetate and 2.5 volumes of 90% ethanol.After precipitation the DNA was pelleted by centrifuging at 12,000 rpmfor 10 minutes. The pellet was washed with 80 μL of 80% ethanol andcentrifuged to dryness using a heated centrifuge. This washing andcentrifugation step was repeated and the DNA thus obtained wasresuspended in 10 μL of sterile water and frozen at −20° C. prior touse.

2. Preparation of Electrocompetent Yeast Cells.

Electrocompetent PichiaPink (pPink) cells were made by inoculating 10 mLof YPD media with a glycerol stock of a genetically engineered Ade2,pep4 knockout pPink yeast strain 2. These cells were grown overnight ina 125 mL baffled flask at 27° C., using a shaker spinning at 270 rpmuntil the OD₆₀₀ of the culture reaches a value of 1.3 units indicatinglog phase growth. This culture was then added to 100 mL of YPD media andallowed to incubate overnight under the same conditions. The OD₆₀₀ waschecked hourly and after a 12 hour incubation period reached a value of1.3 units.

At this time, the culture was transferred to a 500 mL centrifuge tubeand spun for 5 minutes at 4° C. and 5200 rpm. The supernatant wasdecanted and 250 mL of sterile ice cold water was added to resuspend thecells. This wash and centrifugation protocol was repeated twice toensure complete removal of the YPD medium. A final wash was carried outusing 50 mL of sterile ice cold water to suspend the cells followed byrepelleting of the cells at 5200 rpm and removal of the supernatant bydecantation.

To the cell pellet was added 10 mL of sterile ice cold 1M sorbitol. Thesorbitol-cell mixture was then transferred to a sterile 15 mL conicaltube and centrifuged at 5200 rpm. A second wash of the cell pellet using300 μL of sterile ice cold 1M sorbitol was performed prior to usingthese cells for electroporation.

3. Electroporation.

The previously frozen linearized THCA α plasmid DNA or CBDA α plasmidDNA was thawed on ice and 80 μL of the electrocompetent pPink cells wereadded to the tube. This mixture was then transferred to a 0.2 cmelectroporation cuvette and incubated on ice for 5 minutes. The cuvettewas pulsed at 1640V, 200Ω, and 25 μF for a total pulse time ofapproximately 4 ms. Immediately after pulsing 1 mL of YPDS media wasadded to the cuvette and the entire mixture was thoroughly mixed bypipetting. The cuvette was then placed in a 27° C. incubator, withoutshaking, for 2 hours, after which 300 μL was streaked onto fresh PADplates. These plates were incubated at 27° C. for approximately 7-10days to promote cell growth.

4. Screening & Protein Expression

A color based screen was used to identify positive yeast celltransformants. White colonies are indicative of positive expression ofthe gene of interest, whereas red colonies indicate no expression.Accordingly, white colonies were selected from the PAD plates andre-streaked onto fresh PAD plates that were incubated for 3-5 days topromote the growth of individual colonies. A single colony was then usedto inoculate 10 mL of BMGY medium placed in a 125 mL baffled flask thatwas incubated overnight with shaking (270 rpm) at 27° C. The opticaldensity (OD) at 600 nm (OD₆₀₀) was periodically measured for a 1:10diluted sample of the culture.

Incubation was stopped when the inoculum culture attained an OD₆₀₀ of1.2-1.5 units. The inoculum was then transferred to a 50 mL conical tubeand centrifuged at 5200 rpm for 5 minutes to pellet the cells. Afterdecanting the supernatant 1 mL of fresh BMMY medium was added to pellet,following which the tube was covered with an air porous tape thatpermits sterile air exchange and placed in a shaking incubator at 27° C.

After 24 hours a 100 μL of the cell sample was removed to which wasadded 100μL of 40% methanol to induce enzyme production. This methanolicsample was then centrifuged at 12,000 rpm for 5 minutes and thesupernatant and pellet were saved as the T1 sample. A second culturesample was obtained after a 48 hour incubation period and treated withmethanol as described above. After centrifugation, the supernatant andpellet are saved as the T2 sample. At 72 hours the entire incubationmixture is treated with an appropriate volume of 40% methanol. Thesupernatant and pellet obtained from methanolic treatment of the cultureafter a 72 hour incubation period is labeled as T3. The T3 supernatantwas spun through an Amicon 30 kD protein filter and analyzed for proteinusing an SDS-PAGE gel.

Commercial amounts of THCA synthase or CBDA synthase were obtained byscaling up the volume of the culture using a fermentor. Thus, an yeastinoculum was first prepared by contacting 10 mL of BMGY medium in a 125mL baffled flask with a single white colony of yeast. This starterculture was incubated overnight at 27° C. with shaking at 270 rpm toobtain culture with an OD₆₀₀ between 1.2 and 1.5 units. This starterculture was used to inoculate 90 mL of BMGY medium in a 1 L baffledflask. When the OD600 of this culture reached 1.2-1.5 units, theinoculum was transferred to a 500 mL centrifuge bottle and the yeastcells pelleted at 5200 rpm for 5 minutes.

5. Enzymatic Conversion.

The catalytic activity of THCA synthase was measured by incubating 25 μlof the cell free supernatant from a T3 sample with 25 μl of a 1 mg/mlCBGA stock in 200 μl of a 100 mM citrate buffer at pH 4.8 having 10%DMSO for 2 hours at 30° C. The final concentration of CBGA in thereaction mixture was 0.1 mg/ml and the final pH is 5.0. Table 4illustrates the data related to the catalytic activity of THCA synthaseobtained from independent colonies of transformed pPink yeast cells,namely. pPink yeast cells that were transformed using linearized THCAsynthase plasmids. Culture samples that converted greater than 20% ofthe substrate CBGA to product THCA were selected for scale up.

TABLE 4 % Conversion of CBGA to THCA in reaction Sample ID containing0.1 mg/ml CBGA. Spe THC #3 20.6 Spe THC #4 28.7 Spe THC #22 20.6 Spe THC#23 18.7 Pme THC #5 32.5 Pme THC(2) #1 29.1 Pme THC(2) #2A 27.2 PmeTHC(2) #25 31.6 Pme THC(2) #36 27.7 Pme THC(2) #41 32.5 Pme THC(2) #4227.6 Pme THC(2) #46 40.7 Pme THC(2) #51 26.8 Pme THC(3) #1 55.2 PmeTHC(3) #11 35.0 Pme THC(3) #17 69.9 Pme THC(3) #19 36.8 Pme THC(3) #2034.3

6. Cloning Strategy for Generating Multi-Copy GOI Inserts in Vitro.

As described above, transformed cells having 6-10 copies of the gene ofinterest can be used to potentially boost up the production ofheterologous protein. An alternate yeast expression system was used toobtain transformed cells having multiple copies of the gene of interest.The multi-copy Pichia Expression Kit from Invitrogen was used toconstruct a new plasmid that permits yeast cells to be transformed withmultiple copies of the plasmid.

Briefly, the pAO815 vector was used to clone the gene of interest, Thus,the genes for α-CBDA synthase and α-THCA synthase were cut from pPink-HCplasmid using EcoR I and Bam HI restriction enzymes. 100 ng of thepPink-HC vector containing the a-CBDA synthase gene or the α-THCAsynthase gene was incubated with 1 μl of EcoR I buffer, 1 ul of eachrestriction enzyme (10 units/ul) and 1 ul of BSA in a total reactionvolume of 20 ul at 37° C. for 2 hr. Separately, 100 ng of pAO815 vectorwas digested with Eco R I and Bam HI enzymes using the protocoldescribed above.

The digestion mixture is then loaded onto a 0.8% Agarose gel and the DNAfragments were separated from each other by electrophoresis at 95V for 1hr. Bands corresponding the THCA synthase or CBDA synthase genes wereextracted from the gel using the Invitrogen gel extraction kit. Thesegene fragments were ligated to a linearized pAO815 vector using T4 DNAligase and following the ligation protocol from NEB®.

Following ligation, the circular vector containing the gene of interestwas transformed into E. coli Top 10 F″ cells by electroporation at 1500V, 200Ω and 25 μF for 4 ms. The transformed cells were then mixed with250 ul of SOC medium (provided with One Shot® Top 10 Electrocomp™ E.coli from Invitrogen) and plated on a LB-Amp 100 plate at 37 degreesovernight.

After incubation positive colonies were identified using the colony PCRprotocol that relies on 5′ AOX1 and 3′AOX1 primers for performing PCR.Positive colonies containing the gene of interest were grown in liquidLB-Amp 100 media overnight at 37° C. The next day plasmid mini-prepswere done using Invitrogen's fast prep kit and the concentration of theplasmid was analyzed on 0.8% Agarose gel before further amplification.

Once alpha-THCA synthase and alpha-CBDA synthase genes are inserted intopAO815, the recombinant plasmid is divided into 2 batches. The firstbatch was used as a vector in which was inserted a second copy of thegene of interest. The second batch of pAO815 recombinant plasmids wasused for extracting the alpha-THCA synthase or alpha-CBDA synthasegenes. Accordingly, the pAO815 recombinant plasmids used as vectors werefirst digested with Bam HI restriction enzyme following NEB's singledigest protocol. Concurrently, the second batch of pAO815 recombinantplasmids were digested with Bgl II and Bam HI restriction enzymes.

The first and second digestion mixtures were purified using a 0.8%agarose gel followed by extraction of the purified linearized vector andalpha-THCA synthase or alpha-CBDA synthase gene sequences from the gel.Each gene sequence was then ligated with the linearized vector followingNEB's T4 DNA ligase protocol and the vectors containing the gene wereused to transform E. coli Top10 F⁻ cells by electroporation. Thetransformed cells were incubated at 37° C. overnight and then screenedfor the correct gene insert. The above protocol is repeated severaltimes to obtain a multi-copy insert plasmid. After confirming thesequence identity of the gene insert, the multi-copy plasmid waslinearized at the His4 sequence region by restriction enzyme digestionand used to transform competent Pichia pastoris strain G115 (his4, Mut+)cells. The transformed cells were grown on His⁻ plates for screening.Screening was done on His⁻ plates to confirm integration of the plasmidat the His site of the Pichia Pastoris genome. Positive colonies werechosen for methanol induction of protein, and the activity of thesecreted protein was assayed using protocols described above.

Chemical Synthesis A. Synthesis of Geraniol(3,7-Dimethylocta-2,6-dien-1-ol,)

Geraniol was obtained by distillation of palmarosa oil. Palmarosa oil(New Directions Aromatics) was distilled under reduced pressure and thefractions that distil between 139-145° C. and under a reduced pressureof 25 mm Hg were pooled to obtain pure geraniol.

B. Synthesis of Olivetol

Olivetol was synthesized using a published procedure (Focella, A, etal., J. Org. Chem., Vol. 42, No. 21, (1977), p. 3456-3457).

1. Methyl 6-N-Pentyl-2-hydroxy-4-oxo-cyclohex-2-ene-l-carboxylate

To a stirring solution of sodium methoxide (32.4 g, 0.60 mol) anddimethyl malonate (90 g, 0.68 mol) in 230 mL of anhydrous methanol wasadded portion wise 75 g (0.48 mol) of 90% 3-nonen-2-one. The reactionmixture was then refluxed for 3 h under N₂ and allowed to cool to roomtemperature. The solvent was distilled under reduced pressure and theresidue dissolved in 350 mL of water. The slurry of white crystals andthe almost clear solution was extracted thrice with 80 mL of chloroform.The aqueous layer was acidified to pH 4 with concentrated HCl and thewhite precipitate that formed was allowed to stand overnight prior tofiltration. The crystals were dried at 50° C. under high vacuum for 5hours to yield 106.5 g (0.4416 mol) (92%) of methyl6-n-Pentyl-2-hydroxy-4-oxo-cyclohex-2-ene-l-carboxylate (mp 96-98 C).The product was recrystallized using a mixture of petroleum ether:ethylacetate (9:1), and gave 94 g of pure methyl6-n-Pentyl-2-hydroxy-4-oxo-cyclohex-2-ene-l-carboxylate (melting pointof 98-100 C).

2. 1-N-Pentyl-3, 5-dihydroxybenzene (Olivetol).

To a stirring ice-cooled solution of methyl6-N-pentyl-2-hydroxy-4-oxo-cyclohex-2-ene-1-carboxylate (58.4 g, 0.24mol) dissolved in 115 mL dimethylformamide was added dropwise 37.9 g(0.23 mol) of bromine dissolved in 60 mL of dimethylformamide. At theend of the addition (ca. 90 min) the reaction mixture was slowly heatedto 80° C. during which time the evolution of carbon dioxide became quitevigorous.

The reaction was maintained at this temperature until gas evolution hadceased following which the reaction was further heated to 160° C. andheld at this temperature for approximately 10 hours. After heating, thereaction was allowed to cool and the solvent DMF was removed underreduced pressure. The residue thus obtained was treated with water (80mL) and extracted twice with 250 mL of ether. The combined ether layerswere washed with water, then washed with 2×80 mL of a 10% solution ofsodium bisulfate, 2×80 mL of a 10% solution of acetic acid, and thenagain with water.

After drying over anhydrous sodium sulfate the solvent was removed underreduced pressure to give 46.8 g of a viscous oil. The oil was distilledunder reduced pressure to give 30.3 g (0.168 mol) (69.3%) of olivetol asproduct. HPLC analysis indicated 97.5% purity.

C. Synthesis of CBG

CBG was synthesized following the protocol disclosed by Taura et al.,(1996), The Journal of Biological Chemistry, Vol. 271, No. 21, p.17411-17416.

1. Synthesis of2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentyl-benzene-1,3-diol(Cannabigerol (CBG))

Geraniol (3g, 0.0194 mol)and olivetol (2 g, 0.0111 mol) were dissolvedin 400 mL of chloroform containing 80 mg of p-toluenesulfonic acid ascatalyst and the reaction mixture was stirred at room temperature for 12h in the dark. After 12 hours, the reaction mixture was washed withsaturated sodium bicarbonate (400 mL) and then with H₂O (400 mL). Thechloroform layer was concentrated at 40 C under reduced pressure, andthe residue obtained was chromatographed on a 2.0 cm×25 cm silica gelcolumn using benzene (1000 mL) as the eluent to give 1.4 g (0.00442mol)(39.9%) CBG as product.

Alternatively crude CBG was purified as follows. To a 250 mL beaker wasadded 7.25 g crude CBG and 50 mL benzene. The flask was swirled todissolve the CBG and 50 g silica gel was added, along with a stir bar.The solution was stirred overnight, and then poured into a 44 cm×2.75 cmcolumn. The column was eluted with 300 mL benzene. The eluent,approximately 70 mL fractions were assayed for CBG. Fractions 1, 2, and3 (˜230 mL) that contained CBG were combined and the solvent removedunder pressure to give 6.464 g residue containing >80% CBG, having apurity suitable for use in the next synthetic step.

In one embodiment, crude CBG was purified by mixing 7.25 g crude CBGresidue with a slurry of silica gel (50 mL), in a 250 ml Beaker. Thismixture was slowly agitated for 1 hour and then vacuum filtered using afine mesh filter paper. The filter cake was washed with 250 ml benzeneuntil a clear filtrate was obtained. The solvent from the filtrate wasremoved under reduced pressure to give 6.567 g of a residue having >80%CBG.

2. Synthesis of CBG-Acid (CBGA) ?A. Synthesis of MethylmagnesiumCarbonate (MMC)

Methylmagnesium Carbonate (MMC) was synthesized following the protocoldisclosed by Balasubrahmanyam et al., (1973), Organic Synthesis,Collective Volume V, John Wiley & Sons, Inc., p. 439-444.

A dry 2 liter, three necked flask was fitted with a mechanical stirrer,a condenser, and a 1 litre, pressure-equalizing addition funnel, the topof which was fitted with a gas inlet tube. A clean, dry magnesium ribbon(40.0 g, 1.65 mol) was placed in the flask and the system was flushedwith nitrogen prior to the addition of anhydrous methanol (600 mL). Theevolution of hydrogen gas was controlled by cooling the reaction mixtureexternally. When hydrogen evolution had ceased, a slow stream ofnitrogen was passed through the system and the condenser was replaced bya total condensation-partial take-off distillation head. The nitrogenflow was stopped and the bulk of the methanol distilled from thesolution under reduced pressure. Distillation was stopped when stirringof the pasty suspension of magnesium methoxide was no longer practical.The system was again flushed using nitrogen and the outlet from thedistillation head was attached to a small trap containing mineral oil sothat the volume of gas escaping from the reaction system could beestimated.

Anhydrous dimethylformamide (DMF)(700 mL) was added to the reactionflask, and the resulting suspension was stirred vigorously while astream of anhydrous carbon dioxide was passed into the reaction vesselthrough the gas inlet tube attached to the addition funnel. Thedissolution of carbon dioxide was accompanied by an exothermic reactionwith the suspended magnesium methoxide. When no more CO₂ is absorbed,the colorless solution was heated under a slow stream of CO₂ gas untilthe temperature of the liquid distilling reached 140° C., indicatingthat residual methanol had been removed from the reaction mixture. Thereaction mixture was flushed using a slow stream of nitrogen to aid incooling the mixture to room temperature under an inert atmosphere. Thisyielded a solution having 536 mg MMC /mL of DMF.⁸

B. Formation of CBG-A

6-carboxylicacid-2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentyl-benzene-1,3-diol,Cannabigerolic Acid (CBGA) was prepared as follows. To a 10 mL conicalflask was added 1 mL of a DMF solution of MMC. To this solution wasadded 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentyl-benzene-1,3-diol(120 mg, 0.379 mmol). The flask was heated at 120° C. for 1 hour,following which the reaction mixture was dissolved in 100 mL ofchloroform:methanol (2:1) solution. The pH of this solution was adjustedwith dilute HCl to pH 2.0, and then partitioned using 50 mL H₂O.

The organic layer was dried over sodium sulfate and the solvent wasremoved by evaporation. HPLC analysis of the crude reaction showed ˜40%conversion of CBG to CBG-A

Alternatively, 3.16 g (10 mmols)of CBG (or any other neutralcannabinoid), 8.63 g (100 mmols) magnesium methylate and 44 g (1 mol) ofdry ice were sealed in a pressure compatible vessel. The vessel isheated to 50° C., and the temperature held at this value for threehours. Following heating, the vessel is cooled to room temperature andslowly vented. The reaction mixture was dissolved in 100 mL of achloroform: methanol (2:1) solvent. The pH of this solution was adjustedwith dilute HCl to pH 2.0 and this solution was then partitioned using50 mL of H₂O. The organic layer was dried over sodium sulfate and thesolvent was removed by evaporation. HPLC analysis of crude reactionmixture shows ˜85% conversion of CBG to CBG-A using this protocol.

Crude CBG-A was purified by chromatography using a 2.0 cm×25 cm silicagel column. The product was eluted using a mixture of n-hexane:ethylacetate (2:1) (1000 mL), to obtain 45 mg (0.125 mmol)(37.5%) of thedesired product.

Alternatively, ultra high purity CBGA was obtained by chromatographingthe crude using LH-20 lipophilic resin as the medium. 400 g of LH-20Sephadex resin was first swollen using 2 L of DCM:chloroform (4:1)solvent. The swollen resin was gravity packed in a 44 x 2.75 cm column.The column was loaded with 2.1 g of crude CBGA dissolved in a minimumamount of DCM:chloroform (4:1) solvent and eluted with 1.7 L of the samesolvent. 100 mL fractions were collected. The unreacted CBG was elutedas a yellow/orange solution using this solvent system. After the passageof about 1.7 L of this solvent, no more yellow/orange fraction wereobserved and the eluting solvent was changed to 100% acetone to elutethe bound CBGA.

The fractions containing CBGA were pooled and the solvent was removed toobtain 0.52 g CBGA (˜90% recovery). Increasing the volume ofDCM:chloroform (4:1)solvent passed through the column prior to elutingwith acetone, yielded CBGA having purity greater than 99.5%.

Synthesis of CBGV

CBGV was synthesized as follows.

A. Methyl 6-N-Propyl-2-hydroxy-4-oxo-cyclohex-2-ene-l-carboxylate:

Briefly, 3-hepten-2-one (30.1 g, 0.25 mol) was added dropwise to a drymethanolic (125 mL dry MeOH), solution of diethyl malonate (52.016 g,0.323 mol)and sodium methoxide (16.206 g, 0.3 mol). The crude productweighed 46.315 g upon drying at 45° C. overnight in a vacuum oven. Thecrude product was dissolved in petroleum ether (300 mL). After stirring,any undissolved material was filtered from the solution prior to theaddition of ethyl acetate (30 mL), to precipitate CBGV. The precipitatewas filtered and dried overnight at 44° C. in a vacuum oven. A total of33.569 g (0.157 mol) (52.3%) of the desired product was recovered.

B. 1-N-Propyl-3, 5-dihydroxybenzene

A procedure similar to the one described above for the synthesis ofolivetol was used to manufacture the titled compound, except that methyl6-N-propyl-2-hydroxy-4-oxo-cyclohex-2-ene-1-carboxylate was used as thestarting material. Briefly, to a stirring ice cold DMF solution ofmethyl 6-N-propyl-2-hydroxy-4-oxo-cyclohex-2-ene-l-carboxylate was addeda DMF solution of bromine. Following the addition of bromine thereaction mixture was heated to 80° C. Heating was accompanied by thegeneration and release of carbon dioxide gas. After gas evolution hasceased, the temperature of the reaction was increased to 160° C. andheating was continued for 10 hours. The reaction was then cooled and DMFwas removed under reduced pressure. The crude mixture was diluted withwater and subjected to solvent extraction using diethyl ether. Thetitled compound was obtained by removing the ether and distilling theoil that remains.

C. 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propyl-benzene-1,3-diol,(CBGV).

The synthesis of CBGV proceeded by adding p-toluenesulfonic acid to achloroform solution of geraniol and 1-N-Propyl-3,5-dihydroxybenzene.After stirring the reaction at room temperature in the dark for 12hours, water was added to partition the crude product into thechloroform layer. The chloroform layer was then washed with saturatedsodium bicarbonate, dried and the organic solvent removed prior topurification as described above for the synthesis of CBG.

D. 6-carboxylicacid-2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propyl-benzene-1,3-diol(CBGVA).

6-carboxylicacid-2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propyl-benzene-1,3-diol,cannabigerolic Acid (CBGVA) was prepared as follows. Methyl magnesiumcarbonate (MMC) was prepared as described above. To a DMF solution ofMMC in a flask was added2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propyyl-benzene-1,3-diol. Theflask was heated at 120° C. for 1 hour, following which the reactionmixture was dissolved in a 2:1 mixture of chloroform:methanol. The pH ofthis solution was adjusted with dilute HCl to pH 2.0, and the reactionmixture was extracted using H₂O.

The organic layer was dried over sodium sulfate and the solvent wasremoved by evaporation to afford the titled compound CBGVA as the crudeproduct.

Large Scale Enzymatic Production of Cannabinoids

100 ml of a 10 mM sodium phosphate buffer (pH 5.0) were placed in aglass reaction vessel equipped with oxygen gas sparger and a stirrer. Tothis solution 35 g/l of either 2-hydroxypropyl-β-cyclodextrin (HPβCD;Kleptose® HPB), a sulfobutylether β-cyclodextrin sodium salt (SBEβCD;Captisol®), or a randomly methylated β-cyclodextrin (RMβCD) were added.The CD was added in small 5g portions to ensure full dissolution.

2.5 g of a cannabinoid synthase substrate, for example, CBGA or CBGV-Aor a Formula I, II or V compound, were added to the bufferedcyclodextrin solution. The molar ratio of CD to substrate was about 4:1.60 mg of purified synthase were added to the solution and the reactionmixture was incubated at 30° C. for 8 hours. Progress of the reactionwas periodically monitored by HPLC, and using an enzymatic assay todetect and quantify the evolution of hydrogen peroxide.

After 8 hours, greater than 90% of a CBGA substrate was converted toTHCA and CBCA. The ratio of THCA to CBCA was approximately 10:1 at anacidic pH of 5.0. The ratio of the CBC isomers was 5:1.

The aqueous solution was diluted 10:1 with 95% EtOH. This causescyclodextrin to precipitate out leaving the cannabinoids in solution.The cyclodextrin was vacuum filtered, washed with 1 L of 90% EtOH, anddried to permit its reuse in a future reaction. Concentration of theethanolic solution containing the cannabinoids followed suspension ofthe residue in DCM:chlorofrom (4:1) solvent yields ˜25 g crudeorange-yellow residue.

Large Scale Purification of Cannabinoids

Purification of cannabinoids synthesized using a method of thistechnology was accomplished chromatographically using LH-20 lipophilicresin. Briefly, 4000 g of the resin was swollen using 20 L ofDCM:chloroform (4:1). The swollen resin was gravity packed in a 44×27.5cm column. The volume of the swollen resin is ˜1350 mL. The column wasloaded with 25 g crude residue dissolved in a minimum amount of thesolvent and then washed with 4 L DCM:chloroform (4:1) solvent to eluteCBG. No cannabinoid acids were eluted from the column during thiselution.

Gradient elution with a 1:1 to 0:1 DCM:acetone solvent was used to elutethe cannabinoid acids. Each step of the gradient used one column volume(4L) of solvent. CBCA eluted first, followed by CBGA, and then THCA. Thepurity of each cannabinoid was >99.5%.

The pure cannabinoids can further be processed to their neutral or“active” form by heating the acid forms at 90° C. under vacuum.Decarboxylation was quantitative to give the neutral cannabinoid. Ifnecessary, recrystallization can be performed to obtain pharmaceuticalgrade cannabinoids.

What is claimed is:
 1. A system for producing cannabinoids comprising: afermentor holding a cell culture medium comprising genetically modifiedcells producing and secreting a cannabinoid synthase into the medium; abioreactor containing a reaction mixture configured to interact with thecannabinoid synthase to form one or more cannabinoids; and a controlmechanism configured to regulate a property of the medium containing thecannabinoid synthase or to control a property of the reaction mixture;wherein the property of the reaction mixture is one or more of the ionicstrength of the reaction, the pH of the reaction, the temperature of thereaction, or the pressure of the reaction.
 2. The system of claim 1,further comprising a filter configured to separate the cells from themedium containing the cannabinoid synthase produced by the cells.
 3. Thesystem of claim 1, wherein the control mechanism comprises a processingcircuit and a display to control one or more properties of the reactionmixture.
 4. The system of claim 1, wherein the property of the mediumcontaining the cannabinoid synthase is on or more of the oxygen level ofthe medium, the rate of agitation of the medium, the pH of the medium,or the flow rate of the medium into the bioreactor.
 5. The system ofclaim 1, wherein the bioreactor is a column bioreactor containingnickel, and wherein the cannabinoid synthase includes a tag configuredto bond to nickel.
 6. The system of claim 1, wherein the cells are yeastor bacterial cells.
 7. The system of claim 6, wherein the bacterialcells are Escherichia coli cells.
 8. The system of claim 1, wherein thereaction mixture comprises a reagent and a solvent.
 9. The system ofclaim 8, wherein the reagent is cannabigerolic acid (CBGA).
 10. Thesystem of claim 8, wherein the solvent is one or more of dimethylsulfoxide (DMSO), dimethyl formamide (DMF), iso-propoyl alcohol andcyclodextrin.
 11. The system of claim 10, wherein the concentration ofthe solvent in the reaction mixture is between 5% and 30% (w/v).
 12. Thesystem of claim 1, wherein the cells are genetically modified to produceand secrete tetrahydrocannabinolic acid synthase (THCA synthase). 13.The system of claim 12, wherein the system producestetrahydrocannabinolic acid (THCA) and/or cannabichromenic acid (CBCA)in different ratios.
 14. The system of claim 13, wherein thetetrahydrocannabinolic acid (THCA) and/or cannabichromenic acid (CBCA)are eluted and purified.
 15. The system of claim 1, wherein the cellsare genetically modified to produce and secrete cannabidiolic acidsynthase (CBDA synthase).
 16. The system of claim 15, wherein the systemproduces cannabidiolic acid (CBDA) and/or cannabichromenic acid (CBCA)in different ratios.
 17. The system of claim 16, wherein thecannabidiolic acid (CBDA) and/or cannabichromenic acid (CBCA) are elutedand purified.